REGULATION OF GLUTATHIONE SYNTHESIS IN OXIDATIVE STRESS

谷胱甘肽（GSH）是抗氧化防御的重要组成部分。 由醌类产生的氧化应激提高了合成和酶促 γ-谷氨酰半胱氨酸合成酶（GCS）的活性。 这部分地归因 GCS的催化（重）亚基的转录增加。 GCS的调节（轻）亚基的mRNA也增加。 的 GCS酶活性的增加增强了细胞 增加GSH合成，这可能有助于产生更大的抵抗力， 氧化应激从醌类以及环境毒素，如 二氧化氮（NO2）。 本研究的目的是确定调节机制 以及GCS合成增加在氧化应激中的作用。 为这些 研究中，我们将使用体外模型的氧化应激与上皮细胞 来源于大鼠和人肺的细胞系。 具体目标是：（1） 确定负责增加GCS的功能调节元件 通过表征启动子/增强子区域， GCS的催化（重）亚基并剖析功能基序 调节GSCS-HS表达，检查推定的氧化应激反应 GCS-HS区域的启动子/增强子元件 构建体的转录和/或翻译，并确定所述构建体的转录和/或翻译是否与所述构建体的转录和/或翻译相关。 调节（轻）亚基与催化亚基协调。 亚单位 (2)为了确定氧化剂信号转导的机制， 通过表征GCS的增强和持续升高， 参与GCS-HS的氧化应激反应转录因子， 检测细胞H2 O2谷胱甘肽氧化还原状态 (GSH/GSSG）、谷胱甘肽-结合物和谷胱甘肽-蛋白质混合物 二硫化物与GCS重和轻亚基mRNA、蛋白质和 酶活性 (3)确定GSH增加与 合成和增强的抗氧化应激能力， 来自二氧化氮和活性醛的氧化应激产生 类似的增加GSH和GCS观察醌和是否NO2， 反应性醛类和醌类可诱导自身耐受或交叉耐受， 宽容 长期目标是发展药理学 提高GSH的方法，这将提供保护，防止氧化 应力 这可能会扩展到其他病理，如炎症，其中 氧化应激是一个组成部分。

Glutathione (GSH) is an essential component of antioxidant defense. Oxidative stress generated by quinones elevates synthesis and enzymatic activity of gamma-glutamylcysteine synthetase (GCS). This is due, in part to an increase in transcription of the catalytic (heavy) subunit of GCS. The mRNA for the regulatory (light) subunit of GCS is also increased. The increase in GCS enzymatic activity enhances the ability of cells to increase GSH synthesis, which may aid in producing greater resistance to oxidative stress from quinones as well as environmental toxins, such as nitrogen dioxide (NO2). The goal of this investigation is to determine the mechanisms of regulation and roles of the increased synthesis of GCS in oxidative stress. For these studies, we will use in vitro models of oxidant stress with epithelial cell lines derived from rat and human lungs. The specific aims are: (1) To identify the functional regulatory elements responsible for increased GCS in response to quinones by characterizing the promoter/enhancer region of the catalytic (heavy) subunit of GCS and dissecting the functional motif(s) modulating GSCS-HS expression, examining putative oxidative stress response elements of the promoter/enhancer of GCS-HS region using reporter constructs, and determining whether transcription and/or translation of the regulatory (light) subunit is coordinated with that of the catalytic subunit. (2) To determine the mechanism of oxidant signaling involved with the enhancement and sustained elevation of GCS by characterizing the oxidative stress responsive transcription factors involved with GCS-HS and examining the relationship of cellular H2O2 glutathione redox status (GSH/GSSG), glutathione-conjugates, and glutathione-protein mixed disulfides to synthesis of GCS heavy and light subunit mRNAs, proteins and enzymatic activity. (3) Determine the relationship between increased GSH synthesis and enhanced resistance to oxidative stress by examining whether oxidative stress from nitrogen dioxide and reactive aldehydes produce similar increases in GSH and GCS as observed with quinones and whether NO2, reactive aldehydes, and quinones can induce self tolerance or cross tolerance. The long range goal is the development of pharmacological approaches to elevating GSH that will provide protection against oxidative stress. This may extend to other pathologies, such as inflammation, where oxidative stress is a component.